JPH07507769A - Glycosylation-mediated inhibition of factor X - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Glycosylation-mediated inhibition of factor X Technical field The present invention is a drug that inhibits the activation of factor concerns drugs that may be used for prophylaxis. More specifically, the present invention affects the normal glycosylation pattern of factor X, leading to its conversion to its active form. Relating to drugs that reduce The active form of factor X, i.e. factor Xa, is responsible for the formation of thrombin. Thrombin is essential for the formation of blood clots and for the proliferation of smooth muscle cells. It is also an inducing factor.

Technology background Thrombin is a multifunctional protease that controls several important biological reactions. example For example, thrombin is one of the most potent platelet activating factors known. Ru. In addition, thrombin binds fibrinogen to initiate clot formation. It is essential for cleavage into fibrin. These two factors contribute to normal hemostasis. However, in atherosclerotic arteries, blood clots can begin to form. can. Blood clots can cause myocardial infarction, unstable angina a), nonhemorrhagic stroke and vascular A major factor in the pathogenesis of vascular occlusive conditions, such as re-occlusion of coronary arteries after formation or thrombolytic treatment. It is a child. Thrombin is also a strong inducer of smooth muscle cell proliferation and therefore In addition, various proliferative reactions such as restenosis after angioplasty and atherosclerosis induced by transplantation may be involved accordingly. Additionally, thrombin is chemotactic for leukocytes. and may therefore be involved in the inflammatory response (Hoover, R.J., et al. or inhibition of thrombin itself may be useful in preventing and treating thrombosis, limiting restenosis and This suggests that it may be effective in controlling inflammation.

Thrombin is derived from Arg-Th at positions 271-272 of its precursor prothrombin. The r bond and the Arg-1ie bond at positions 320-321 are proteolytically cleaved. formed as a result of This activity occurs on the cell membrane surface of platelets, monocytes and epithelial cells. catalyzed by the assembled prothrombinase complex. The complex is factor Xa (serine protease), factor Va (cofactor), calcium ions and acidic Contains lipid surfaces. Factor Xa is the active form of its precursor factor X. X factor is liver It is secreted as a 58 kd precursor from It is converted to active factor Xa in Precursor of factor X and Va, factor ■ The circulation level is known to be on the order of 10-'M. Corresponding active type■ Levels of factor a and factor Xa have never been measured.

The complete amino acid sequences of human factors X and Xa are known, and Davie.

Described by E., W. (Hemostasis and Thro mbos engineering, 2nd edition, edited by R, W, Coleman et al. (1987) 250 page). Factor X is calcium ion binding, gamma carboquine glutamyl (Gl Contains a>, depends on vitamins, is a member of the blood-clotting glycoprotein family . This family includes factors VII and IX, prothrombin, protein The mature protein of factor X has a 40-residue brepro leader sequence. , which is removed during intracellular processing and secretion. Xa The factor It is cleaved into a double-stranded form by removal of the three amino acid residues (RKR) in between. lastly, Double-chain Factor It produces a 39-residue light chain and a 254-residue heavy chain, which are converted to factor Xa. these are , connected through one disulfide bond between position 128 of the light chain and position 108 of the heavy chain. I'm looking forward to it. ! The I chain contains a Gla region and an epidermal growth factor-like region. Professional Tease activity is present in the heavy chain, with histidine at position 12 and asparagine at position 88. and contains serine at position 185.

Research was also conducted on the false factor X, and its amino acid sequence was reported (Tit ani, ni, Proc, Natl, Acad, Sci, USA (1975 )72:3082-3086). The activation peptide of bovine factor X has a length of 51 It is an amino acid residue and contains approximately equal amounts of polypeptide and carbohydrate (Discip io.

R, G, et al., Biochemistry (1977) 16:5253).

There are two known routes to activate double-stranded Factor X in vivo. activity conversion must occur before the protease is incorporated into the prothrombinase complex. (Steinberg, M. et al., Hemostasis and T. hrombos is, edited by Coleman, R.W., et al. (1987) J.

B, Lippencott, Ph1ladelphia, PA, p, 112 ). In the intrinsic pathway, factor IXa assembled on the cell surface, Factor X is opened by the tenase J complex containing lucium ions. cleaved, releasing a 52 amino acid activation peptide. In the extrinsic route, tissue on the membrane Cleavage is catalyzed by Factor VIIa, which is bound to Factor VI. Russell Kusari In vitro cleavage using proteases such as those contained in snake venom The X factor may be converted to the Xa factor by This protease is Disci pio.

As described by R, G. et al. (Biochemistry (1977) 6:5253). It is listed.

There are also variants of factor X that are differentially activated in the extrinsic and intrinsic pathways. (Fair, D, S, et al., J, Cl1n Invest (1979) For example, the X factor "Vorarlberg" is With respect to the extrinsic pathway it has 15% of the activity of normal factor X and with respect to the intrinsic pathway has 75% of the activity of normal factor X (Watzke, H, H, et al. , J. Biol, C1jin, (1990) Dandan Mutual 11982-11989) .

Does factor Xa play an essential function in forming thrombin from its precursor? By appropriately manipulating factor X/factor Xa, platelet coagulation and clot formation It is clear that thrombin activity could be suppressed in

The activity of factor Xa, which affects the conversion of prothrombin to thrombin, Depends on its incorporation into the rhombinase complex. Therefore, one aspect of this operation is The approach was to prevent factor Xa from participating in the thrombinase complex. . Prothrombinase complex (for the conversion of prothrombin to thrombin) Studies have been conducted on the formation of factor Xa, which acts 278,000 times faster than soluble (Nesh e im, H, E, et al, J, Biol, Chem, (1979) λ54:10952). These studies demonstrate that active site-specific inhibition Factor, dansyl glutamyl glycyl arginyl (DEGR) chloromethyl I've been using ketones. This covalently attaches a fluorescent acceptor group to Factor Xa. This obstacle Factor Xa treated with harmful factors loses protease activity, but is identical to factor Xa. is incorporated into the brothrombinase complex with a stoichiometry of 2.7 (Nesheim, M. E., J. Biol. Chem. (1 981) λdandan: 6537-6540: Skogen, W, F et al., J, Bi ol, C animals 12953-12961).

Other attempts at factor Xa inhibition currently include tissue factor pathway inhibitors (tissue factor pathway inhibitors). It is called factor pathway 1 inhibitor (TFPI). lipoprotein-associated coagulation inhibitors (lipoprotein-associated coagulation inhibitors) ated coagulation 1nhibitor (LACI) Girard, s, J.

et al., Nature (1989) 338:518; Girard ), T. J. et al., 5science (1990) 2481421)), Hill Yu. Antistatin (Dunviddie, C.T., et al.) JBiol, Chem, (1989): 26416694), and mites Originated from TAP (Taxman).

Shira, 5science (1990) 2=+8:593) Ru. Alternatively, drugs that inhibit vitamin-dependent Gla-converting enzymes, e.g. Phosphorus is also used. These attempts have been plagued by lack of specificity, high volume requirements, and harmful None of them have been successful due to side effects and long delays in efficacy.

Another method disclosed in PCT International Publication No. 91106337 involves the activation of factor Xa. Modification of the sexual site maintains the ability to form the prothrombinase complex while increasing the enzyme activity. is inhibiting.

The method of the present invention inhibits the conversion of factor X to its active form, factor Xa. The aims. Specifically, this mode of inhibition is due to the glycosylation associated with factor By manipulation of residues.

The glycosylation pattern of bovine, but not human, factor X has not been studied in detail. Ru. The bovine factor X stalwart has N-linked glycosylation at residue 36; Has 〇-linked glycosylation at the 000th group (Mizuochi) , T, et al., J, inhibition o1. Chell, (1980) 255: 352 6-3531). The glycosylation pattern of human factor X is unknown; The activation peptide has N-linked glycosylation potential at positions 39 and 49. It has two functional parts (Davie, E, W,, rhemeo). Stasis and Thrombosis J, 2nd edition, edited by R.W. Coleman, (1987) 2 50 pages). In addition, the related epidermal growth factor-like domain of bovine factor IX contains Since the existence of serine-linked sugar chains has been reported (Base, S.

et al., J. Biol, Chew, (1990) 265: 1858-1 861); Does the EGF-like domain of human factor X light chain also contain 〇-linked sugar chains? Maybe.

Therefore, the present invention inhibits the conversion of factor Provides another method of inhibiting the formation of enzyme complexes.

Disclosure of invention The present invention is effective in reducing thrombus formation and thrombin-induced restenosis and inflammation. effective therapeutic agents useful in preventing and treating the progression of other pathological conditions of the vascular system. provide. This means that in Western society, the occurrence of blood clots is the main cause of death. , and the problem of re-narrowing with increasing application of angioplasty and other vascular procedures. It is important because it is increasing. The therapeutic substance of the present invention inhibits the production of human factor Xa. can be harmed or diminished.

These therapeutic agents are particularly useful for preventing the rapid onset of thrombosis. this It is used to prevent thrombus formation in coronary arteries in patients with angina pectoris at rest, and to prevent relapse after thrombolytic treatment. Prevention of thrombosis, including prevention of thrombosis during complex angioplasty procedures. These therapeutic agents are To prevent smooth muscle cell proliferation following angioplasty or other vascular surgery. It will also be useful. The therapeutic agent of the present invention is based on the current standard treatment using heparin. (Hanson, RlS, et al., Proc, Natl, ca d, Sci. (1988) 85: 3184). provide In one embodiment, the compound of the invention is a lectin, which is human Binds specifically to the carbohydrate moiety present in factor and inhibits its conversion to factor Xa . In another embodiment, the compounds of the invention themselves alter the glycosylation structure. acquire the ability.

In one aspect, the present invention provides human factor X and galactose or N-acetyl Has the ability to bind to terminal sialic acid linked α(2-6) to lugalactosamine Activation of human factor X by contacting with specific carbohydrate-binding reagents Regarding prevention methods. Hereinafter, these target glycosyl groups are each labeled as SA(2-6) aGal and SA (2-6) Abbreviated as aGalNAc. The reagent used is rS A/Gal/GalNAcJ binding reagent. This carbohydrate binding reagent is When exposed to human factor X under appropriate temperature and pH conditions, human factor or is effective in preventing conversion to human factor Xa through the endogenous pathway. . Thus, in another aspect, the present invention provides Concerning the complex.

In another aspect, the invention provides for carbohydrate moieties forming derivatives together with Factor X. cleavage of factor X or prevention of noarylation of factor Concerning the factors that cause sex.

Another aspect of the invention includes carbohydrate binding reagents or carbohydrate opening agents useful as therapeutic agents. Thrombin-initiated pharmaceutical compositions of fission reagents and those compositions can be used to Also included are methods of preventing or treating thrombosis or other pathological conditions that may occur.

Brief description of the drawing Figures IA and IB show bovine factor X and human factor This paper presents the results of investigating the effects of these factors on the coagulation activity when treated with The invention relates to the blocking of specific carbohydrate residues or cleavage of carbohydrate moieties of human Factor X. is the conversion of human factor X to its active form, which is essential for the formation of the prothrombinase complex. This is based on the discovery that it inhibits More specifically, the glycosylation part , a sialic acid residue is attached to a galactose or N-acetylgalactosamine residue by α(2 -6) At the attached non-reducing end, a vine reagent (SA /Ga1/Ga1NAc binding reagent) can perform this inhibition. these The reagents include various lectins (for example, Sambucus c us nigra) agglutinin (SNA, elderberry bark lectin) and and some mammalian lectins, as well as other small molecules (e.g. peptides) that are known or can be shown to be peptides. This point , peptides derived from factor IXa/■a or tissue factor ■a are particularly useful. be.

Other important SA/Gal/Ga1NAc binding reagents specifically target this target It is an antibody that is reactive. Such antibodies may contain any of these trisaccharide moieties. , keyhole green clam hemocyanin, human serum albumin, bovine serum albumin Immunogenicity of min, diphtheria toxoid, rotavirus VP6 carrier protein, etc. Conveniently manufactured by conjugation with an imparting carrier to render it immunogenic Can be done. suitable for attaching carbohydrates to this type of immunogenic carrier. techniques, e.g., converting the carbonyl moiety of a carbohydrate to an available amino group on the carrier. Techniques for conjugation followed by reduction are well known in the art.

then combined with an immunogenic carrier on its own or as a larger carbohydrate The trisaccharide moieties obtained are administered to appropriate subjects via standard immunization protocols. to induce antibody production. The effectiveness of this protocol is determined by the serum For example, an immunogen, or preferably a suitable trisaccharide moiety thereof, is combined with an antigen, i.e. Monitored by performing standard immunoassays using specific binding partners. can be done. Such immunoassays, e.g. radioimmunoassays, Enzyme-linked immunoassays, fluorescent type immunoassays, etc. are well known in the art. It is well known. When sufficient titer is obtained, immunoglobulin purification The subsequent antiserum can be used directly (or, alternatively, in pancreatic or peripheral cells of the immunized animal). monoclonalization of these antibodies immunospecific for trisaccharide moieties using blood leukocytes. It can be manufactured in a single format. Generally, antibody-producing cells are hybridomas, etc. Immortalization in the form of is also now standard practice.

In addition to these SA/Ga1/GaINAc binding reagents, factor Antibodies (including monoclonal antibodies) that bind to the carbohydrate moiety of Xa will inhibit the formation of the factor.

In another approach, the glycosylated form of the sialic acid terminal residues of Factor Interfering with activation is also an effective way to obtain factor X that resists activation.

In one approach, inhibition of alpha(2-6) sialyltransferase This objective can be achieved by administering the agent to the organism. That is, The organism will produce Factor X lacking essential substituents necessary for its activation. cormorant. Since internal glycans must serve as acceptors for sialic acid residues, Therefore, a variation on this approach is commonly used to catalyze the glycosylation of Factor X. Inhibitors of sferases may also be effective. Inhibitors of glycosylated factor Organisms to be effective in changing all means (repe r to i re) supplied to i.e. characterized by an excess of Factor Xa or For conditions that would benefit from a reduction in the levels of progenitors, these inhibitors are considered interfering agents. At the same time, it acts as a preventive medicine. Such inhibitors include Amphomycin, castanospermine, 2,3-dehydro-2-deoxy-N -acetyl-neuraminic acid, 1-deoxymannojirimycin hydrochloride, 1-deo Xynodymycin, N-methyl-1-deoxynodymycin, Swainso and tunicamycin.

In another approach, the conversion of factor X to factor Xa clearly requires Soluble analogues of the SA/Gal/GaI NAc sequence were synthesized for competing essential disaccharide residues. It can be used as a mixture. Such a competitor would otherwise be to Factor Xa is introduced under conditions such that the conversion of F to Factor Xa is achieved.

That is, in vitro, soluble analogs are Introduced with components of the sexual pathway. For whole living organisms, soluble analogs administered and disrupt this conversion in 5 situ. Soluble analogs of factor SA (2-6) aGal or SA (2 -6) A low molecular weight molecule with a spatial and charge profile similar to αGa1NAc residue. be. Such soluble analogs include SA(2-6)aGal and SA (2-6) αGa1NAc itself, trisaccharides and trisaccharide analogs or there are other complex structures that exhibit these characteristics.

In yet another approach, the conversion of factor X to factor Xa is from factor X to factor Xa. Reagents that remove glycosylated moieties, thereby rendering them immune to activation. Therefore, it can be inhibited. A suitable such reagent is Neuraminidar. There are certain specific glycosidases that remove enzymes and sialic acid residues.

A preferred method of inhibiting the conversion of factor X to factor Xa is glycosylation of factor SA (2-6) aGal and SA (2-6) αGalNA in the side chain Utilizes a binding agent that forms a complex with the c residue. Generally, the method of the invention useful as SA/Gal/GalNAc binding reagents for use in The reagent contains a trisaccharide moiety (or can be identified using a simple specific binding assay using Wear. Such assays are performed by methods similar to immunoassays.

For example, in a typical protocol, the trisaccharide moiety to be targeted is The bound support is bound to a support and the bound support is treated with a candidate reagent. Next, the processed The carrier is washed to remove unbound candidate compound. Any residue remaining attached to the support A co-compound is a label that specifically binds to the compound and is itself detectable (e.g. appropriate means such as additional moieties labeled with e.g. radioactive or enzymatic labels). Detected by. That is, candidate SA/Ga l/Ga 1NAc binding reagent Compounds can be quickly screened for efficacy with such a simple and easy protocol. -ing can be done.

In the method of the present invention, SAα(2-6)Gal identified by the above method and/or SAα (2-6) Ga l NAc binding reagent, whose conversion is inhibited. contact with the X factor to be treated. Such contact may result in the release of exogenous or endogenous Either route results in a complex that is resistant to conversion to the active form. the contact using an excess of the reagent, preferably a 2- to 10-fold molar excess, to form a complex. The test is carried out at appropriate temperature, pH 1 and salt conditions. Generally, such contact in the presence of physiological saline and physiological pH for 4 hours to overnight at 4°C to room temperature. To be carried out in the country.

The SA/Gal/Ga1NAc binding compound of the present invention, which is a lectin, is a natural source. and, in some circumstances, by recombinant techniques. Manufactured. Alternatively, it comprises a SA/Ga l/Ga lNAc binding domain. Synthetic peptides may be used in place of full-length lectins. These peptides are Synthesized using standard solid phase or solution phase peptide synthesis techniques that are known and known in the art. It is practically commercially available.

The SA/Ga1/Ga1NAc binding reagent of the present invention and the inhibition of factor X activation The above-mentioned selective methods that can be harmful include treatments with complications of thrombosis as well as etiological factors. This is useful in situations involving thrombin generation. These situations include: Arterial thrombosis, e.g. coronary and peripheral angiogenesis and athelial thrombosis. rectomy) and during vascular bypass procedures (peripheral and coronary). and subsequent unstable (i.e. resting) angina and acute vascular occlusion, myocardial infarction. re-occlusion after thrombolytic treatment, thrombotic stroke (stroke in evolution), and vasculitis. This includes thrombosis (Kawasaki's disease). Venous thrombosis, for example, deep in the lower extremities Venous thrombosis, pulmonary artery embolism, renal vein, hepatic vein, caval vein thrombosis, and cavernous vein side Also included are situations involving thrombosis. Other target conditions include widespread activation of the coagulation system. , for example, Sevenos with scattered intravascular coagulation, in other consolidations. Disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, and rare situations of unknown etiology ( anticoagulant (anticoagulant).

SA/Ga l/Gal NAc binding reagent of the present invention and inhibition of activation of factor X Selective methods also apply in the preparation of blood products or samples, as well as in organ transfusions. Cardiopulmonary bypass in harvested organs in transplantation and related treatment of skin recipients. It is also useful as an anticoagulant and anti-inflammatory agent. These reagents and methods are In particular, in-dwelling vascular devices (e.g. catheters, grafts, patches) Useful in immediate release form.

Thrombosis can also be caused by directly or indirectly causing smooth muscle cell proliferation. , after vascular interventions such as angioplasty, atherectomy or arterial endarterectomy. It also plays a role in restenosis, and the reagents of the invention also treat this condition. It is also useful in this regard.

Adult respiratory distress syndrome (ARDS) is a disease in which the prothrombinic endothelium becomes inflammatory and proliferative. It is considered to be an "endotoxin" disease that is likely to exist together with sexual components; Ming's reagents and methods are also useful in the treatment of ARDS.

lectins or their peptide binding domains, or antibodies or their or other peptide-based substances, the therapeutic reagents of the invention are fragments of Typically injected using conventional excipients for protein or peptide administration. by, e.g., Remington's PharIllaceutic al　5sciences,　Mack」Publish■ Company, Latest Edition, Easton, PA. Formulated for administration. lectins or other peptides for antithrombotic effects. tide or protein, preferably by infusion, and preferably by intravenous infusion administered systemically. The level of dosage will depend on the condition of the subject and the specific Depends on a number of factors, including aspects of the process. However, the appropriate dosage range is continuous Levels range from 1 to 50 mg per patient per infusion dose. For injection, tap Proteins are transferred to liquid media such as Hank's solution, Ringer's solution, etc. er), dextrose solution, and various buffers.

Additional excipients such as stabilizers can also be used.

In addition to injection, the lectin or pond therapeutic reagents of the present invention can be administered via suppositories, orally, or nasally. Systemically via transmucosal administration, including internal sprays, and by slow-release formulations. It can be administered to Additional formulation techniques include encapsulation such as liposomes. Includes formulations.

In addition to their utility as therapeutic agents, the reagents of the invention can be used to generate polyclonal antisera. or by producing cells that can be fused with an immune partner. can be used to obtain a source of monoclonal antibodies specific for these reagents. Wear. These antibodies serve as diagnostic tools for monitoring treatments using the reagents of the invention. It is useful as

SA/Gal/Ga of the present invention for inhibiting the conversion of factor X to factor Xa The ability of 1NAc binding reagents or other candidates to produce the enzymatic activity properties of factor X subjected to putative activation conditions of extrinsic or intrinsic pathways related to can be studied by measuring the effect of a candidate inhibitor on the ability of a factor to Wear. Therefore, the presence of the generated factor Xa is due to the presence of chromozyme Bonyl-D-norloinlugulin-arginine-4-nitranilide acete (Boehringer Mannheim) as measured by the hydrolytic kinetics of hydrolysis. Factor X was added at 0.2 nM to produce the putative Factor Factor IXa of 5. 8 nM factor VIII a (Cutter hemoph ilia concentrate), an excess of phospholipids in the mM range, and and activated with 5 to 10 mM calcium ions. Activation of factor X Incubate in saline for approximately 10 minutes at 37°C, and then This is done by detecting factor Xa formed by hydrolysis of mozyme X.

Activation of factor VIII to VIIIa was banks), D, C,, J Rial Cheva (1990) 1785 4-17858.

To determine the effect of SA/Gal/Ga1NAc binding reagent, factor X and Candidate binding reagents were first added, followed by calcium, magnesium and manganese ions, respectively. Braid at low temperature for 4 to 24 hours in buffered saline containing approximately 1mM each. Incubate. Similarly, candidate inhibitory enzymes that remove glyconylation of factor Breincubate with this factor before adding components of the endogenous pathway. SA/ To test the inhibitory ability of competitive soluble analogs of Gal/Ga1NAc residues , the soluble analog is added along with the components of the pathway. However, these inhibitions Since the efficacy of the agent depends on the formation of factor X in living objects, This assay method is not suitable for those reagents that are inhibitors of glycosylation. do not have.

Due to the effect on the extrinsic pathway, the conversion of factor X to factor Xa is reduced from 0.5 to 1 nM factor VII, tissue factor (thromboplastin C), excess phospholipids and performed using 5 to 10 mM calcium ions (for intrinsic pathway activation) as described).

The above methods are SA/Ga l/Ga lNAc binding reagents or For example, a candidate compound that inhibits the formation of factor Xa will show inhibition of factor X activation. This is an example of how to confirm. These assays are based on glycosidase, or SA /Ga l/Ga l NAc binding reagent as well as removing glycosylated units Other reagents that can be used to determine their efficiency in inhibiting factor used. The assay set described above is used as described above and includes candidate Glycondase or other glycolylation removal enzyme, or SA/Ga1/Ga1 A soluble version of the SA/Gal/GalNAc sequence containing an NAc binding reagent and Includes brain incubations with analogues.

The following examples are intended to illustrate the invention and are intended to illustrate the invention. It is not intended to limit clarity.

Example 1 Reserved ability of lectins with different binding specificities to complex with human and bovine factor X Human and bovine factor X of lectins with different binding specificities were tested in and the ability to complex with factor Xa using glycan identification kits (First Choice, Base -Ringer Mannheim). Hands performed according to manufacturer's instructions In this order, Factor X or Factor Xa is first applied to a nitrocellulose or nylon membrane. specificity of the lectin immobilized on top and labeled with digokingenin from the kit. Binding was tested using an anti-ngoxigenin antibody conjugated to alkaline phosphate. I put it out. Table 1 summarizes the results obtained using the various lectins of the kit.

cow human Ga1anthus n1valis high mannose chain ---1-agglutinin (GN ^) Peanut agglutinin (PN^) Ga1 (β1-3) GalNAc, --- (Ilo SA) Datura straIlionium Ga1(β1-4)GalNAc; --+ -Agglutinin (DSA), GlcNAc Sambucus nigra SA(α2-6)Gal--+-agglutinin ( SNA) SA(a2-6)Gal2-6)GalNAc amurensi s SA(α2-3)Gal + -10-Agglutinin (MAA) Therefore, DSA, SNA and MAA bind to human factor X, and bovine factor Bound only by MAA. Both activated forms of these lectins did not combine with

human or bovine factor X (1 μM) and candidate compound lectin (5 μM), respectively. at 4°C in buffered saline containing 1mM Ca"2, Mg+2 and Mn" ions. It was left to incubate overnight. IXa, VII Activated as described above with Factor Ia, phospholipids and calcium ions. free The factor Xa was measured using a Chromozyme X probe as described above. either Lectin also does not inhibit the activation of bovine factor X, and only SNA inhibits the activation of human factor X. inhibited. The degree of inhibition was 41%.

Bovine or human factor Incubate in the same manner and then add factor VIIa, tissue factor, phospholipids and and activated with calcium ions as described above. Generation of factor Xa is as described above. It was decided. None of the lectins inhibited the activation of bovine factor X, and only SNA Again, activation of human factor X was inhibited (67% inhibition).

Human factor X (1 μM) was incubated at 4°C with SNA at concentrations ranging from 0 to 20 μM. and incubated overnight. Aliquots were tested using activators of the intrinsic and extrinsic pathways. Measurements were made using the same method as described in Examples 2 and 3 above. Bovine factor X as a control showed mild inhibition only at the highest concentrations of SNA.

In activation of the intrinsic pathway using IXa/Vlla, 1 μM SNA or higher At a concentration of 1.5 μM, inhibition was 40%. , increased to approximately 47% at 20 μM.

In the extrinsic activation pathway, virtually no inhibition was obtained with 0.5 μM SNA. However, 1 μM SNA showed approximately 27% inhibition, and 20 μM SNA showed 80% inhibition. Increased to inhibition.

Glyconidases with different specificities were tested according to the manufacturer's instructions. Treatment of human and bovine factor X by treating factor X with recdase I used it to Digestion was mostly carried out at 37°C at night, but Neuraminid. The enzyme was completely digested in a shorter period of time. The reaction mixture was purified by SDS/PAG. Factor Protein mobility shifts were determined relative to untreated Factor X. bovine factor x or for any of the human factor X, for any of the glycosidases, for the light chain It showed no shift in mobility. However, some of the glycosidases tested , resulted in changes in the mobility of the heavyweights as described in Table 2 below.

The effect of deglycosylation on activity was tested in several ways. The first method is Standard coagulation analysis method for glycosylated substances - activated partial thromboplastin Tested with time analysis (APTT) and prothrombin time analysis (PT).

Analyzes were performed using standard procedures, using factor X-deficient human plasma and automated coagulation analysis. I used a container. The results of these analyzes are also shown in Table 2.

Additionally, glycosidase-processed proteins can be converted into factor IXa/factor VIIIa complexes or or activated factor Xa using VT factor Ia/tissue factor complex. was detected using Chromozyme X. These results were compared to bovine factor The X factor is shown in Figure IA and Figure IB, respectively.

Table 2 Human factor X, bovine factor X An, C, APTT PT AH, C, APTT PT (kd) (kd) Control -100100---1001000-Glyconodase 6.2 61 108 3.3 116 106 Endoglycondase HO49600881 06 Neuraminidase 4.5 12 8 4.5 4 2N-gluconodase F 7 148 37 7 97 102 Table 2 shows the addition of H chain mobility software. and the APTT and PT coagulation test results as a percentage of control activity. show. Only neuraminidase had a dramatic effect on reducing coagulant activity. but, Endoglycosidase H was partially effective on human factor X.

Figures IA and IB show chromozyme X for bovine factor X and human factor The results of each activity by the extrinsic or intrinsic pathway are further analyzed.

Only neuraminidase has the ability to substantially reduce activity by either pathway ( Yes, consistent with the results of coagulation activity in Table 2.

Run X Human X Continuation of front page (81) Designated countries EP (AT, BE, CH, DE.

DK, ES, FR, GB, GR, IE, IT, LU, ~IC, NL, PT, SE ), 0A (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, S N.T.D.

TG), AT, AU, BB, BG, BR, CA, CH.

DE, DK, ES, Fl, GB, HU, JP, KP, KR, LK, LU, N iG, MN, MW, NL, No, NZ, PL, PT, RO, RU, S D, SE, US, VN

Claims (30)

[Claims] 1. α2-6 binding of human factor X to galactose or galactosamine residues A reagent that can specifically bind to sialic acid residues (SA/Gal/GalNAc binding reagent ) in an amount and for a time sufficient to form a complex with factor X. , a method of inhibiting the conversion of human factor X to human factor Xa. 2. Claim 1, wherein the SA/Gal/GalNAc binding reagent is a lectin. How to put it on. 3. Claim 2, wherein the lectin is Sambucus coaglutinin (SNA). The method described in. 4. Claim 1, wherein the SA/Gal/GalNAc binding reagent is an antibody. Method. 5. Claims wherein the SA/Gal/GalNAc binding reagent is a mammalian lectin The method described in Section 1. 6. The SA/Gal/GalNAc binding reagent is linked to factor IXa/VIIIa or tissue. 2. The method of claim 1, wherein the peptide is derived from Factor/Factor VIIa. 7. A complex consisting of a SA/Gal/GalNAc binding reagent and human factor X. 8. Claim 7, wherein the SA/Gal/GalNAc binding reagent is a lectin. complex. 9. Claim 8, wherein the lectin is Sambucus coaglutinin (SNA). The complex described in. 10. Claim 7, wherein the SA/Gal/GalNAc binding reagent is an antibody. complex. 11. Claims wherein the SA/Gal/GalNAc binding reagent is a mammalian lectin A complex according to paragraph 7. 12. The SA/Gal/GalNAc binding reagent is linked to factor IXa/VIIIa or The complex according to claim 7, which is a peptide derived from tissue factor/VlIa factor. body. 13. The active ingredient SA/Gal/GalNAc binding reagent is pharmaceutically acceptable. Pharmaceutical compositions useful for inhibiting the conversion of factor X to factor Xa . 14. Claim 13, wherein the SA/Gal/GalNAc binding reagent is a lectin. The composition described in . 15. Claim 1 wherein the lectin is Sambucus coaglutinin (SNA). Composition according to item 4. 16. Claim 13, wherein the SA/Gal/GalNAc binding reagent is an antibody. composition. 17. Claims wherein the SA/Gal/GalNAc binding reagent is a mammalian lectin A composition according to paragraph 13. 18. The SA/Gal/GalNAc binding reagent is linked to factor IXa/VIIIa or The composition according to claim 13, which is a peptide derived from tissue factor/factor VIIa. A product. 19. Effective amounts of SA/Gal/GalNAc binding for human subjects in need of treatment. Prevention of thrombosis in a human subject comprising administering a reagent or pharmaceutical composition thereof or how to treat it. 20. Effective amounts of SA/Gal/GalNAc binding for human subjects in need of treatment. Preventing inflammation in a human subject by administering a reagent or pharmaceutical composition thereof. or how to treat it. 21. Effective amounts of SA/Gal/GalNAc binding for human subjects in need of treatment. Preventing restenosis in a human subject comprising administering a reagent or pharmaceutical composition thereof or how to treat it. 22. Effective amounts of SA/Gal/GalNAc binding for human subjects in need of treatment. Transplant complications in human subjects that involve administering reagents or pharmaceutical compositions thereof. How to prevent or treat. 23. human factor X and glycosylated moieties from the factor Contacting the reagent in an amount and time sufficient to open the glycosylated moiety. A method of inhibiting the conversion of human factor X to human factor Xa. 24. The method according to claim 23, wherein the glycosidase is neuraminidase. . 25. Can bind human factor X and the glycosylated portion of the activation peptide of factor contacting the reagent in an amount and for a time sufficient to bind the peptide. , a method of inhibiting the conversion of human factor X to human factor Xa. 26. Soluble species of human factor X and at least one SA/Gal/GalNAc the soluble analog, the contacting comprising contacting the soluble analog with a Human factor Xa conversion of human factor X under conditions that affect the conversion of factor X to factor Xa How to inhibit conversion into children. 27. 27. The condition according to claim 26, wherein the condition is an extrinsic or intrinsic pathway condition. Method. 28. Claims that contact is made by administering a soluble analog to a living organism The method according to item 26. 29. A living organism comprising administering to an organism an effective amount of a glycosylation inhibitor. A method of inhibiting the conversion of human factor X to human factor Xa in an organism. 30. Claim 29, wherein the inhibitor inhibits sialyltransferase. the method of.